Summary Influenza virus, a member of the orthomyxovirus family, has three different types that infect humans (A, B, and C). Types A and B produce annual epidemics, and type A influenza, which resides asymptomatically in birds, can cause pandemic infections in humans. In addition to common flu-like symptoms (e.g., fever, chills, malaise, muscle pain), in susceptible populations influenza can induce potentially fatal secondary complications including bronchitis, pneumonia, and acute respiratory distress. Worldwide, influenza infection can cause up to 5 million severe cases and 500,000 deaths per year. Influenza is also a considerable economic burden with an estimated annual cost of $87.1 billion in the U.S. alone. Existing antivirals can treat influenza but must be given within 48 hours of infection to be effective. Additionally, many flu strains have developed complete resistance to older antivirals. Therefore, the best method to dampen the spread of disease is preventative vaccination. Even though traditional influenza vaccines have been on the market since the 1930s, they have several limitations: dependence on egg-based technology to grow the virus, limited worldwide vaccine availability, decreased efficacy in the elderly, and lack of cross-reactivity with various influenza strains. Each year, based on predictions from the WHO and CDC recommendations, manufacturers formulate the flu vaccine using just three to four killed or attenuated viruses. Since 2004, the influenza vaccine has been 40% effective on average with some years showing very low effectiveness of only 10%. This poor effectiveness is likely due to the targeted viral strains undergoing antigenic drift and/or shift. Development of a ?universal flu vaccine? using safe recombinant (i.e., not egg-based) subunit antigens could potentially help overcome the drawbacks of the current influenza vaccine strategy. In order to develop a universal vaccine the immune system must generate a response against conserved epitopes that are found on many different strains of influenza. These epitopes include the stalk domain of HA, the matrix protein 2 ectodomain, and other proteins that induce CD8 T cell activity against the virus. In order to generate an effective immune response against these subunit antigens, an adjuvant must be co- administered to provide protection. We have preliminary data demonstrating that a novel adjuvant encapsulated in a proprietary microparticle (MP) can induce a T-helper 1 cellular immune response, and using a universal influenza antigen it can help to provide superior protection against a lethal challenge compared to other commercially available adjuvants, as well as cross-reactive antibodies across different viral subtypes. Based on this preliminary data we propose scaling up the fabrication of these MPs. The data collected from this proposal would allow us to manufacture these MPs on an industrial scale. We will validate that the MP-adjuvanted subunit antigen can protect against influenza infection and generate an immune response similar to what was fabricated in the bench-top setting. We will also study the sterilization of these MPs, which is an important step in the potential manufacturing of the proposed MP platform. The data generated in this proposal will be crucial for a Phase II application.